The unremarkable alveolar epithelial glycocalyx: a thorium dioxide-based electron microscopic comparison after heparinase or pneumolysin treatment.

Recent investigations analyzed in depth the biochemical and biophysical properties of the endothelial glycocalyx. In comparison, this complex cell-covering structure is largely understudied in alveolar epithelial cells. To better characterize the alveolar glycocalyx ultrastructure, unaffected versus injured human lung tissue explants and mouse lungs were analyzed by transmission electron microscopy. Lung tissue was treated with either heparinase (HEP), known to shed glycocalyx components, or pneumolysin (PLY), the exotoxin of Streptococcus pneumoniae not investigated for structural glycocalyx effects so far. Cationic colloidal thorium dioxide (cThO2) particles were used for glycocalyx glycosaminoglycan visualization. The level of cThO2 particles orthogonal to apical cell membranes (≙ stained glycosaminoglycan height) of alveolar epithelial type I (AEI) and type II (AEII) cells was stereologically measured. In addition, cThO2 particle density was studied by dual-axis electron tomography (≙ stained glycosaminoglycan density in three dimensions). For untreated samples, the average cThO2 particle level was ≈ 18 nm for human AEI, ≈ 17 nm for mouse AEI, ≈ 44 nm for human AEII and ≈ 35 nm for mouse AEII. Both treatments, HEP and PLY, resulted in a significant reduction of cThO2 particle levels on human and mouse AEI and AEII. Moreover, a HEP- and PLY-associated reduction in cThO2 particle density was observed. The present study provides quantitative data on the differential glycocalyx distribution on AEI and AEII based on cThO2 and demonstrates alveolar glycocalyx shedding in response to HEP or PLY resulting in a structural reduction in both glycosaminoglycan height and density. Future studies should elucidate the underlying alveolar epithelial cell type-specific distribution of glycocalyx subcomponents for better functional understanding.

Platelet extracellular vesicles mediate transfusion-related acute lung injury by imbalancing the sphingolipid rheostat.

Transfusion-related acute lung injury (TRALI) is a hazardous transfusion complication with an associated mortality of 5% to 15%. We previously showed that stored (5 days) but not fresh platelets (1 day) cause TRALI via ceramide-mediated endothelial barrier dysfunction. As biological ceramides are hydrophobic, extracellular vesicles (EVs) may be required to shuttle these sphingolipids from platelets to endothelial cells. Adding to complexity, EV formation in turn requires ceramide. We hypothesized that ceramide-dependent EV formation from stored platelets and EV-dependent sphingolipid shuttling induces TRALI. EVs formed during storage of murine platelets were enumerated, characterized for sphingolipids, and applied in a murine TRALI model in vivo and for endothelial barrier assessment in vitro. Five-day EVs were more abundant, had higher long-chain ceramide (C16:0, C18:0, C20:0), and lower sphingosine-1-phosphate (S1P) content than 1-day EVs. Transfusion of 5-day, but not 1-day, EVs induced characteristic signs of lung injury in vivo and endothelial barrier disruption in vitro. Inhibition or supplementation of ceramide-forming sphingomyelinase reduced or enhanced the formation of EVs, respectively, but did not alter the injuriousness per individual EV. Barrier failure was attenuated when EVs were abundant in or supplemented with S1P. Stored human platelet 4-day EVs were more numerous compared with 2-day EVs, contained more long-chain ceramide and less S1P, and caused more endothelial cell barrier leak. Hence, platelet-derived EVs become more numerous and more injurious (more long-chain ceramide, less S1P) during storage. Blockade of sphingomyelinase, EV elimination, or supplementation of S1P during platelet storage may present promising strategies for TRALI prevention.

Sodium-coupled neutral amino acid transporter SNAT2 counteracts cardiogenic pulmonary edema by driving alveolar fluid clearance.

The constant transport of ions across the alveolar epithelial barrier regulates alveolar fluid homeostasis. Dysregulation or inhibition of Na+ transport causes fluid accumulation in the distal airspaces resulting in impaired gas exchange and respiratory failure. Previous studies have primarily focused on the critical role of amiloride-sensitive epithelial sodium channel (ENaC) in alveolar fluid clearance (AFC), yet activation of ENaC failed to attenuate pulmonary edema in clinical trials. Since 40% of AFC is amiloride-insensitive, Na+ channels/transporters other than ENaC such as Na+-coupled neutral amino acid transporters (SNATs) may provide novel therapeutic targets. Here, we identified a key role for SNAT2 (SLC38A2) in AFC and pulmonary edema resolution. In isolated perfused mouse and rat lungs, pharmacological inhibition of SNATs by HgCl2 and α-methylaminoisobutyric acid (MeAIB) impaired AFC. Quantitative RT-PCR identified SNAT2 as the highest expressed System A transporter in pulmonary epithelial cells. Pharmacological inhibition or siRNA-mediated knockdown of SNAT2 reduced transport of l-alanine across pulmonary epithelial cells. Homozygous Slc38a2-/- mice were subviable and died shortly after birth with severe cyanosis. Isolated lungs of Slc38a2+/- mice developed higher wet-to-dry weight ratios (W/D) as compared to wild type (WT) in response to hydrostatic stress. Similarly, W/D ratios were increased in Slc38a2+/- mice as compared to controls in an acid-induced lung injury model. Our results identify SNAT2 as a functional transporter for Na+ and neutral amino acids in pulmonary epithelial cells with a relevant role in AFC and the resolution of lung edema. Activation of SNAT2 may provide a new therapeutic strategy to counteract and/or reverse pulmonary edema.

A transmembrane C-terminal fragment of syndecan-1 is generated by the metalloproteinase ADAM17 and promotes lung epithelial tumor cell migration and lung metastasis formation.

Syndecan-1 is a heparan sulfate proteoglycan expressed by endothelial and epithelial cells and involved in wound healing and tumor growth. Surface-expressed syndecan-1 undergoes proteolytic shedding leading to the release of the soluble N-terminal ectodomain from a transmembrane C-terminal fragment (tCTF). We show that the disintegrin and metalloproteinase (ADAM) 17 generates a syndecan-1 tCTF, which can then undergo further intra-membrane proteolysis by γ-secretase. Scratch-induced wound closure of cultured lung epithelial A549 tumor cells associates with increased syndecan-1 cleavage as evidenced by the release of shed syndecan-1 ectodomain and enhanced generation of the tCTF. Both wound closure and the associated syndecan-1 shedding can be suppressed by inhibition of ADAM family proteases. Cell proliferation, migration and invasion into matrigel as well as several signaling pathways implicated in these responses are suppressed by silencing of syndecan-1. These defects of syndecan-1 deficient cells can be overcome by overexpression of syndecan-1 tCTF or a corresponding tCTF of syndecan-4 but not by overexpression of a tCTF lacking the transmembrane domain. Finally, lung metastasis formation of A549 cells in SCID mice was found to be dependent on syndecan-1, and the presence of syndecan-1 tCTF was sufficient for this activity. Thus, the syndecan-1 tCTF by itself is capable of mediating critical syndecan-1-dependent functions in cell proliferation, migration, invasion and metastasis formation and therefore can replace full length syndecan-1 in the situation of increased syndecan-1 shedding during cell migration and tumor formation.

Road traffic noise impacts sleep continuity in suburban residents: Exposure-response quantification of noise-induced awakenings from vehicle pass-bys at night.

Nocturnal traffic noise has been associated with adverse health outcomes in exposed residents. Precise quantification of traffic noise effects on sleep is thus of great importance. Here we establish an exposure-response relationship for the awakening probability due to intermittent road traffic noise in suburban residents. We conducted a field study in residential areas where road traffic was the dominant noise source, and noise events were attributable to separate vehicle pass-bys. Forty healthy participants underwent polysomnography for five consecutive nights at their homes. A total of 11,003 road traffic noises derived from simultaneous acoustic measurements at the sleepers' ears were included in an event-related analysis of awakenings. Logistic regression analysis revealed that the awakening probability due to road traffic noise increased with the maximum sound pressure level (SPL) and the maximum slope of the increasing SPL of a vehicle pass-by, as well as the age of the exposed individual. Compared to sleep stage 2, the awakening probability was higher in rapid eye movement sleep (REMS) and lower in slow wave sleep (SWS). The protective effect of both stage 2 and SWS against awakenings decreased with age, whereas no age-dependent change was observed for REMS. When adjusting for other contributing factors, the probability of a noise-induced awakening ranged from 0% at a maximum SPL of 27.1 dB(A) to 2.0% at 70 dB(A). Road traffic noise at night - even in suburban areas with moderate traffic density - negatively impacts residents' sleep continuity. Exposure-response quantification for traffic noise-induced awakenings may serve as a basis for noise protection efforts by regulators and policy makers.

Loss of endothelial CFTR drives barrier failure and edema formation in lung infection and can be targeted by CFTR potentiation.

Pneumonia is the most common cause of the acute respiratory distress syndrome (ARDS). Here, we identified loss of endothelial cystic fibrosis transmembrane conductance regulator (CFTR) as an important pathomechanism leading to lung barrier failure in pneumonia-induced ARDS. CFTR was down-regulated after Streptococcus pneumoniae infection ex vivo or in vivo in human or murine lung tissue, respectively. Analysis of isolated perfused rat lungs revealed that CFTR inhibition increased endothelial permeability in parallel with intracellular chloride ion and calcium ion concentrations ([Cl-]i and [Ca2+]i). Inhibition of the chloride ion-sensitive with-no-lysine kinase 1 (WNK1) protein with tyrphostin 47 or WNK463 replicated the effect of CFTR inhibition on endothelial permeability and endothelial [Ca2+]i, whereas WNK1 activation by temozolomide attenuated it. Endothelial [Ca2+]i transients and permeability in response to inhibition of either CFTR or WNK1 were prevented by inhibition of the cation channel transient receptor potential vanilloid 4 (TRPV4). Mice deficient in Trpv4 (Trpv4-/-) developed less lung edema and protein leak than their wild-type littermates after infection with S. pneumoniae. The CFTR potentiator ivacaftor prevented lung CFTR loss, edema, and protein leak after S. pneumoniae infection in wild-type mice. In conclusion, lung infection caused loss of CFTR that promoted lung edema formation through intracellular chloride ion accumulation, inhibition of WNK1, and subsequent disinhibition of TRPV4, resulting in endothelial calcium ion influx and vascular barrier failure. Ivacaftor prevented CFTR loss in the lungs of mice with pneumonia and may, therefore, represent a possible therapeutic strategy in people suffering from ARDS due to severe pneumonia.

Short-Term Annoyance Due to Night-Time Road, Railway, and Air Traffic Noise: Role of the Noise Source, the Acoustical Metric, and Non-Acoustical Factors.

Field studies on traffic noise-induced annoyance have predominantly used estimated outside noise levels. We intended to complement existing knowledge with exposure-response relationships that are based on precise indoor noise measurements. Acoustic recordings inside the bedrooms of nightly road traffic and annoyance ratings in the following morning were obtained from 40 suburban residents (mean age 29.1 years ± 11.7; 26 females). We derived exposure-response functions for the probability to be "annoyed at least a little" (%LA). Further analyses compared data from the current study with those from two earlier studies on railway and aircraft noise. Annoyance increased with the number of traffic events and the equivalent sound pressure level. The inclusion of non-acoustical factors (such as assessment of road transport) improved the prediction considerably. When comparing the different traffic noise sources, %LA was higher for road than for air traffic at a given LAeq,night, but higher for road and railway than for air traffic at a given number of noise events. Acoustical as well as non-acoustical factors impact short-term annoyance induced by road, railway, and air traffic. Annoyance varies across noise sources, which may be due to differences in acoustical characteristics or in the temporal noise distribution throughout the night.

Influence of transient pressure changes on speech intelligibility: Implications for next-generation train travel.

High-speed trains are operated in increasingly complex railway networks and continual improvement of driver assistance systems is necessary to maintain safety. Speech offers the opportunity to provide information to the driver without disrupting visual attention. However, it is not known whether the transient pressure changes inside trains passing through tunnels interfere with speech intelligibility. Our primary goal was to test whether the most severe pressure variations occurring in high-speed trains (25 hPa in 2 s) affect speech intelligibility in individuals with normal hearing ability and secondly whether a potential effect would depend on the direction of the pressure change. A cross-over design was used to compare speech intelligibility, measured with the monosyllable word test by Wallenberg and Kollmeier, in steady ambient pressure versus subsequent to pressure events, both realised in a pressure chamber. Since data for a power calculation did not exist, we conducted a pilot study with 20 participants to estimate variance of intra-individual differences. The upper 80% confidence limit guided sample size of the main campaign, which was performed with 72 participants to identify a 10% difference while limiting alpha (5%) and beta error (10%). On average, a participant understood 0.7 fewer words following a pressure change event compared to listening in steady ambient pressure. However, this intra-individual differences varied strongly between participants, standard deviation (SD) ± 4.5 words, resulting in a negligible effect size of 0.1 and the Wilcoxon signed rank test (Z = -1.26; p = 0.21) did not distinguish it from chance. When comparing decreasing and increasing pressure events an average of 0.2 fewer words were understood (± 3.9 SD). The most severe pressure changes expected to occur in high-speed trains passing through tunnels do not interfere with speech intelligibility and are in itself not a risk factor for loss of verbal information transmission.

Cytokine-Regulation of Na+-K+-Cl- Cotransporter 1 and Cystic Fibrosis Transmembrane Conductance Regulator-Potential Role in Pulmonary Inflammation and Edema Formation.

Pulmonary edema, a major complication of lung injury and inflammation, is defined as accumulation of extravascular fluid in the lungs leading to impaired diffusion of respiratory gases. Lung fluid balance across the alveolar epithelial barrier protects the distal airspace from excess fluid accumulation and is mainly regulated by active sodium transport and Cl- absorption. Increased hydrostatic pressure as seen in cardiogenic edema or increased vascular permeability as present in inflammatory lung diseases such as the acute respiratory distress syndrome (ARDS) causes a reversal of transepithelial fluid transport resulting in the formation of pulmonary edema. The basolateral expressed Na+-K+-2Cl- cotransporter 1 (NKCC1) and the apical Cl- channel cystic fibrosis transmembrane conductance regulator (CFTR) are considered to be critically involved in the pathogenesis of pulmonary edema and have also been implicated in the inflammatory response in ARDS. Expression and function of both NKCC1 and CFTR can be modulated by released cytokines; however, the relevance of this modulation in the context of ARDS and pulmonary edema is so far unclear. Here, we review the existing literature on the regulation of NKCC1 and CFTR by cytokines, and-based on the known involvement of NKCC1 and CFTR in lung edema and inflammation-speculate on the role of cytokine-dependent NKCC1/CFTR regulation for the pathogenesis and potential treatment of pulmonary inflammation and edema formation.

A cytoplasmic C-terminal fragment of Syndecan-1 is generated by sequential proteolysis and antagonizes Syndecan-1 dependent lung tumor cell migration.

Syndecan-1 is a surface expressed heparan sulphate proteoglycan, which is upregulated by several tumor types and involved in tumor cell migration and metastasis. Syndecan-1 is shed from the cell surface and the remaining transmembrane fragment undergoes intramembrane proteolysis by γ-secretase. We here show that this generates a cytoplasmic C-terminal fragment (cCTF). In epithelial lung tumor A549 cells the endogenously produced cCTF accumulated when its proteasomal degradation was blocked with bortezomib and this accumulation was prevented by γ-secretase inhibition. Overexpression of the cCTF suppressed migration and invasion of A549 cells. This inhibitory effect was only seen when endogenous Syndecan-1 was present, but not in Syndecan-1 deficient cells. Further, overexpression of Syndecan-1 cCTF increased the basal activation of Src kinase, focal adhesion kinase (FAK) and Rho GTPase. This was associated with increased adhesion to fibronectin and collagen G and an increased recruitment of paxillin to focal adhesions. Moreover, lung tumor formation of A549 cells in mice was reduced by overexpression of Syndecan-1 cCTF. Finally, delivery of a synthetic peptide corresponding to the Syndecan-1 cCTF suppressed A549 cell migration and increased basal phosphorylation of Src and FAK. Our data indicate that the Syndecan-1 cCTF antagonizes Syndecan-1 dependent tumor cell migration in vitro and in vivo by dysregulating proadhesive signaling pathways and suggest that the cCTF can be used as an inhibitory peptide.